Project Summary Immunization with antigen in the presence of agonists for both a Toll Like Receptor (TLR) and CD40 (combined TLR/CD40 immunization) elicits a vigorous expansion of antigen-specific CD8+ T cells that is exponentially greater than the response elicited by either agonist alone. Not only is the primary immune response to this vaccination robust, it also forms long lived, CD8+ T cell memory that can protect against future infectious challenge even in the absence of CD4+ T cells. This has been recently verified in non- human primates, where the vaccine produced responses exponentially stronger than responses to typical viral vectors. Given the potency and clinical potential for this vaccine adjuvant platform, it is critical that we understand its molecular and cellular mechanistic underpinnings. We recently made the surprising discovery that T cell responses to this, and related, vaccinations were unexpectedly and completely dependent on two cytokines (IL-27 and IL-15) and two transcription factors (Tbet and Eomes). This was unexpected because the loss of any one of these factors essentially ablates the response to the vaccine but has little to no effect on the response to live virus or bacteria. Thus, the rules behind robust subunit vaccine-elicited immunity appear to be substantially different than those guiding infectious responses. This proposal will use cutting edge methods and approaches to fully understand the nature of this difference and will test i) how IL-27 and IL-15 influence downstream transcriptional and cellular factors, ii) how Tbet and Eomes expression subsequently program T cell activation and expansion, and iii) how these factors mediate T-DC and T-T interactions during the earliest events of T cell activation after combined adjuvant vaccination.